The main objective of this research project is to gain insight into the structure and function of the Na, K-ATPase. The physiological characteristics of the Na,K-ATPase will be studied using the alpha and beta polypeptides synthesized from an in vitro rabbit reticulocyte translation system or a yeast expression system. Expression of the subunits separately and together will determine the role of each individual subunit in supporting Na,K- ATPase function. Once activity is obtained, specific deletions and modifications in the amino acid sequence of the subunits will be used to determine the role of the specific domains in mediating transport. The kinetic parameters of Na,k and ATP will be determined, and the characteristics of the partial pump reactions studied. The transport characteristics of the alpha-subunit isoforms will also be investigated. The characterization of transport in the normal and specifically modified enzymes should provide information about the mechanism of transport of the Na,K-ATPase. The cardiac glycosides, which specifically inhibit the Na,K-ATPase, are used clinically in the treatment of conjestive heart failure. The interaction of the cardiac glycoside, ouabain, with the Na,K-ATPase will be studied using the Na,K- ATPase from the ouabain-resistant rat. To determine the mechanism of ouabain-resistance, hybrid proteins consisting of proteins of the rat and human polypeptides will be made. Also, specific amino acids which may be involved in the mechanism of ouabain-resistance will be modified. The interaction of ouabain with these hybrid and modified Na,K-ATPases, and their influence on transport, may give insight into the interaction and mechanism of ouabain inhibition. To investigate the physiological role of the Na,K-ATPase isoforms during development, in situ hybridization of whole body sections of fetal rats will be performed. These results may provide information on the functional roles of Na,K- ATPase isoforms.